PROJECT SUMMARY ? PROJECT 2 Project 2 made considerable progress over the past four years in defining the influence of several histone methyltransferases and demethylases, acting in prefrontal cortex (PFC) neurons, in controlling depression- related phenomena. A major finding from this work is that genome regulation is often ?punctate? as opposed to continuous and affects certain clusters along the genome. Consistent with this notion is our evidence that depression-related epigenetic regulation partly bypasses the linear genome and affects the 3D structure of chromatin. One of the most dramatically affected genomic regions is the Pcdh locus, which expresses numerous isoforms of protocadherins, strongly implicated in neuronal growth and synapse formation, but poorly understood in adult brain, let alone depression. Importantly, the Pcdh locus also displays robust regulation in the Center's genome-wide datasets, including dramatic induction in PFC of mouse models and depressed humans, effects seen in males and females, and Pcdh genes are among the most highly induced in PFC by early life stress. We will now further characterize the influence of the 3D genome, with particular attention to Pcdh, in depression in several key ways. We will study how manipulation of specific proteins which control 3D chromatin structure in PFC neurons influence depression-related behavioral abnormalities. We will use gene-editing tools, with the Chromatin and Gene Analysis Core, to modify the Pcdh locus and study directly its influence in depression models. This is a required approach, since the large number of Pcdh genes and compensatory changes in other isoforms when a single isoform is manipulated has made it difficult to characterize protocadherin function in adult brain. We will also map how chronic stress alters the 3D genome in PFC neurons, both focusing on Pcdh as well as the global genome, with parallel studies of depressed humans performed in Project 4. Finally, we will employ a novel method to retrospectively map how the 3D genome is altered by early life stress when examining adult mice based on their susceptibility to subsequent stress. Together, these studies will reveal how Pcdh genes influence depression-related phenomena and provide fundamentally new information about the 3D genome in adult neurons.